Amphiregulin Regulates Proliferation and Differentiation of ATDC5 Cells through PI3K/AKT Signaling Pathway

doi:10.13701/j.cnki.kqyxyj.2024.07.006

Abstract

Abstract: Objective: To explore the impact of amphiregulin (Areg) on the proliferation and osteogenic differentiation of ATDC5 cells. Methods: Quantitative real time polymerase chain reaction (qRT-PCR) was used to assess Areg expression in ATDC5 cells. The expression of Areg in tibial growth plates underwent analysis via immunohistochemical staining. Lentivirus-mediated Areg overexpression or knockdown was conducted in ATDC5 cells, and the impact on cell proliferation was evaluated using CCK-8 assay. qRT-PCR and Western blot analyses evaluated the impact of Areg on osteogenic differentiation in ATDC5 cells on markers associated with 7-day, such as Sox9 and collagentype Ⅱ alpha1chain (Col2α1) or 14-day, such as collagentype Ⅹ alpha1chain (ColⅩα1), osteopontin (OPN), and Runt-related transcription factor 2 (Runx2). Cartilage matrix formation was assessed using staining techniques. Western blot analysis was conducted 7 days after initiating ATDC5 cells osteogenic differentiation to assess the phosphorylation of the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) pathway. The phosphorylation of PI3K/AKT was attenuated following the administration of the p-AKT inhibitor MK2206. Results: Areg expression persisted throughout the osteogenic differentiation of ATDC5 cells, peaking on day 7. Areg was highly expressed in chondrocytes of tibial growth plates. Furthermore, Areg overexpression significantly enhanced cell viability, chondrogenic differentiation and extracellular matrix formation. While the opposite effect was observed when Areg was knockdown. Areg overexpression activated PI3K/AKT pathway, and the addition of p-AKT inhibitor MK2206 reversed the activation. Conclusion: Areg promotes the proliferation of ATDC5 cells, and partially stimulates the osteogenic differentiation of ATDC5 cells via the PI3K/AKT pathway.

Key words: chondrocyte, cell proliferation, osteogenic differentiation, PI3K/AKT signaling pathway

WANG Xinyi, WANG Xinru, WANG Shuo, WANG Jiawei. Amphiregulin Regulates Proliferation and Differentiation of ATDC5 Cells through PI3K/AKT Signaling Pathway[J]. Journal of Oral Science Research, 2024, 40(7): 599-604.

References

[1] Scotti C, Piccinini E, Takizawa H, et al. Engineering of a functional bone organ through endochondral ossification [J]. Proc Natl Acad Sci U S A, 2013, 110(10):3997-4002.
[2] Zaiss DMW, Gause WC, Osborne LC, et al. Emerging functions of amphiregulin in orchestrating immunity, inflammation, and tissue repair [J]. Immunity, 2015, 42(2):216-226.
[3] Qin L, Tamasi J, Raggatt L, et al. Amphiregulin is a novel growth factor involved in normal bone development and in the cellular response to parathyroid hormone stimulation [J]. J Biol Chem, 2005, 280(5):3974-3981.
[4] Kimura H, Schubert D. Schwannoma-derived growth factor promotes the neuronal differentiation and survival of PC12 cells [J]. J Cell Biol, 1992, 116(3):777-783.
[5] Pasic L, Eisinger-Mathason TS, Velayudhan BT, et al. Sustained activation of the HER1-ERK1/2-RSK signaling pathway controls myoepithelial cell fate in human mammary tissue [J]. Genes Dev, 2011, 25(15):1641-1653.
[6] Ulici V, Hoenselaar KD, Gillespie JR, et al. The PI3K pathway regulates endochondral bone growth through control of hypertrophic chondrocyte differentiation [J]. BMC Dev Biol, 2008, 8:40.
[7] Kita K, Kimura T, Nakamura N, et al. PI3K/Akt signaling as a key regulatory pathway for chondrocyte terminal differentiation [J]. Genes Cells, 2008, 13(8):839-850.
[8] Li G, Wang L, Jiang Y, et al. Upregulation of Akt signaling enhances femoral fracture healing by accelerating atrophic quadriceps recovery [J]. Biochim Biophys Acta Mol Basis Dis, 2017, 1863(11):2848-2861.
[9] Shukunami C, Ishizeki K, Atsumi T, et al. Cellular hypertrophy and calcification of embryonal carcinoma-derived chondrogenic cell line ATDC5 in vitro [J]. J Bone Miner Res, 1997, 12(8):1174-1188.
[10] Kirimoto A, Takagi Y, Ohya K, et al. Effects of retinoic acid on the differentiation of chondrogenic progenitor cells, ATDC5 [J]. J Med Dent Sci, 2005, 52(3):153-162.
[11] Wuelling M, Vortkamp A. Transcriptional networks controlling chondrocyte proliferation and differentiation during endochondral ossification [J]. Pediatr Nephrol, 2010, 25(4):625-631.
[12] Berasain C, García-Trevijano ER, Castillo J, et al. Novel role for amphiregulin in protection from liver injury [J]. J Biol Chem, 2005, 280(19):19012-19020.
[13] Hsu D, Fukata M, Hernandez YG, et al. Toll-like receptor 4 differentially regulates epidermal growth factor-related growth factors in response to intestinal mucosal injury [J]. Lab Invest, 2010, 90(9):1295-1305.
[14] 杜小颖,丁威薇,陈悦,等.IGF-1通过PI3K/AKT信号通路促进牙髓细胞增殖及碱性磷酸酶活性的研究[J].口腔医学研究,2017,33(5):538-541.
[15] Zhang H, Chen X, Xue P, et al. FN1 promotes chondrocyte differentiation and collagen production via TGF-β/PI3K/Akt pathway in mice with femoral fracture [J]. Gene, 2021, 769:145253.